www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 28), pp: 19469-19480 Research Paper: Immunology Identification of neoepitopes recognized by tumor-infiltrating lymphocytes (TILs) from patients with glioma Davide Valentini1, Martin Rao2, Qingda Meng2, Anna von Landenberg2, Jiri Bartek Jr3,4, Georges Sinclair4, Georgia Paraschoudi2, Elke Jäger5, Inti Harvey-Peredo4, Ernest Dodoo4 and Markus Maeurer1,2,5 1Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital Huddinge, Stockholm, Sweden 2Therapeutic Immunology Unit (TIM), Department of Laboratory Medicine (LABMED), Karolinska Institutet, Stockholm, Sweden 3Department of Neurosurgery, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark 4Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden 5 Krankenhaus Nordwest, Division of Oncology and Hematology, Frankfurt, Germany Correspondence to: Markus Maeurer, email: [email protected] Keywords: tumor-infiltrating lymphocytes; immunotherapy; neoepitopes; glioblastoma; interferon gamma; Immunology Received: September 11, 2017 Accepted: February 24, 2018 Published: April 13, 2018 Copyright: Valentini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of ‘private’ somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients’ responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies. INTRODUCTION identified based on the amino acid sequence derived from antigens recognized by a specific population of T cells The identification of T-cell epitopes provided from defined by immune reactivity, i.e. cytokine production, host-derived targets is crucial in developing personalized usually measured as antigen-induced interferon gamma treatments for patients with advanced cancer. Epitopes are (IFN-γ) responses. This process does not only allow to www.oncotarget.com 19469 Oncotarget identify new cancer-specific targets, but also to identify way by which clinically relevant TIL responses can be T-cell receptors (TCR) that can be used in the development gauged and tailored for personalized immunotherapy. of immunotherapies. Several approaches of epitope identification are RESULTS currently in use. Mass spectrometry-based sequencing of peptides eluted from tumor cell-derived human leukocyte Clinical characteristics of patients antigen (HLA) molecules aims to decipher naturally processed and presented peptides, although there may Five patients were diagnosed with GBM (WHO be limits concerning sensitivity in this approach [1, 2]. grade IV tumor of the central nervous system), involving Screening of cDNA libraries encoding tumor-associated different anatomical compartments of the brain. A antigens (TAAs) has been used extensively; this approach description of the clinical characteristic of the patients is is labor-intensive and minimally effective, as larger provided in Table 1. transcripts and weakly expressed transcripts cannot be cloned easily. Furthermore, this method also eliminates TIL reactivity to wildtype and mutated peptides identification of some mutated antigens since it does not cover post-transcriptional modifications of protein A schematic representation of the entire process antigens [2]. The peptide-based screening approach workflow pertaining to the current study is presented identifies mutations through whole-exome sequencing in Figure 1. Immunoreactivity of the TILs from five followed by in-silico analysis, based on an algorithm different patients with GBM to private epitopes (patient- that predicts the peptide binding capacity of the major specific peptides generated from the whole-exome histocompatibility complex (MHC)-peptide complex [3, sequencing data) were evaluated in TIL cultures. A heat 4]. The drawback of this approach is that the antigen- map depicting cytokine responses (IFN-γ, TNF-α and/or receptor interaction is based on an algorithm that predicts IL-17A) following a 7-day in vitro stimulation assay with the MHC binding capacity, and is not thoroughly studied the wildtype as well as mutated peptide sequences was for MHC class II alleles or infrequent HLA alleles [2, 5]. generated for each patient (Figure 2). In Table 2, we show The discovery of epitopes using this approach is only as how many mutations from GBM lesions (5 patients) good as the in-silico prediction itself, which is limited are recognized by TILs based on IFN-γ production. for MHC class II alleles and for a number of MHC The amount of cytokine produced by TILs, considered class I molecules. A novel and revolutionary method as a readout for target-specific immunoreactivity, is the tandem minigene (TMG) approach, where the is presented based on a scale of low to high cytokine patients’ ‘private’ mutations are identified using whole- concentration in the culture supernatants (Figure 2). A exome sequencing in order to subsequently construct more comprehensive list, detailing individual cytokine a personalized library of gene sequences encoding concentration values (in pg/10e5 TIL/7 days) per peptide mutated epitopes – or neoepitopes. In the TMG setting, for the five patients with GBM is also presented in only a small portion of the gene around the mutation is Supplementary Table 1. Mutated and wildtype peptides synthesized, and its reaction with autologous T cells is alike were able to activate CD4+ and CD8+ TILs to tested to identify whether the predicted neoepitopes are produce a single cytokine or a combination of the three naturally processed and presented to the immune system cytokines measured (IFN-γ, TNF-α and/or IL-17A). [6] based on the assumption that the surrogate antigen- We noticed that a positive cytokine response by TILs presenting cells process and present the neoepitopes in a to the peptides tested (mutated and/or wildtype) was similar fashion to tumor cells. dominated by IFN-γ production. TILs from patients In the present study, we have used a combination of GBM-A and GBM-D appeared to exhibit the strongest whole-exome sequencing and individual peptide responses overall peptide-driven Th1 immune responses (IFN-γ to screen for immune reactivity among in vitro-expanded and/or TNF-α). Several of these peptides (mutated and/ tumor-infiltrating lymphocytes (TILs) from patients or wildtype) also resulted in IL-17A production by TILs. with glioblastoma (GBM) [7], the most lethal form of Patients GBM-A and GBM-B displayed only weak to brain cancer marked by a 5-year survival rate of under very low IL-17A responses to any of the peptides tested. 3% [8, 9]. The approach described here allows for direct While TILs from patient GBM-B produced loading of 15-mer peptides onto MHC class II molecules, measurable amounts of IFN-γ to twelve out of eighteen or by uptake, processing and subsequent presentation peptide pairs (mutated and/or wildtype) and did not mount of potential epitopes by MHC class I or MHC class II a strong IL-17A response to any of the peptides tested, molecules on surrogate antigen-presenting cells (APCs) TILs from patient GBM-E produced substantial amounts to enable T-cell engagement and activation. We report of IFN-γ in response to ten out of eleven mutated peptides. a method that helps the identification of antigenic (neo) Interestingly, TILs from patient GBM-E also produced a epitopes that are presented either directly or indirectly (via combination of IFN-γ and IL-17A in response to three processing) to T cells in the tumor microenvironment, a mutated peptides. In terms of peptide-driven TNF-α www.oncotarget.com 19470 Oncotarget Table 1: Clinical characteristics of patients Patient ID Age Sex Diagnosis Grade Tumor localization GBM-A 67 M GBM IV Thalamus GBM-B 62 F GBM IV Temporal GBM-C 63 F GBM IV Temporal GBM-D 65 M GBM IV Frontal GBM-E 67 F GBM IV Parietal